In cells, external stimulation with a ligand is transmitted into cells via a receptor. In the case of chemokine receptors, for example, it is known that leukocyte chemotaxis, which is the fundamental function in inflammation and immune responses, is strictly controlled by attractors serving as agonists (Documents 1 to 4). More specifically speaking, a chemokine prototype CCL2 (also known as MCAF and MCP-1) was found as a macrophage attractant mediated by a receptor CCR2 (Documents 5 to 8). Moreover, chemokines CCL3, 4 and 5 are known as agonists for CCR5.
It has been considered that intracellular signal transduction of a receptor depends on the G protein switching mechanism. Concerning chemokines, there have been identified more than 50 types of chemokines and 20 types of G protein-coupled receptors (GPCRs) as the chemokine-chemokine receptor family. Each chemokine receptor has a strict chemokine-specificity and shows an expression pattern restricted to leukocyte subtype (Documents 9 to 11).
Now, the relationships between the intracellular signal transduction of receptors and diseases will be discussed. In the case of the chemokine receptor CCR2, for example, the receptor CCR2 is a 7-transmembrane G protein-coupled receptor known as occurring in monocytes, macrophages, lymphocytes, endothelial cells, smooth muscular cells and so on and its agonist CCL2 acts as an attractant via interaction with the receptor CCR2 (Document 12). It is considered that the CCL2-CCR2 pathway participates in the causes of atherosclerosis (Documents 13 and 14), chronic glomerulonephritis (Document 15), multiple sclerosis (Documents 16 and 17) and other chronic inflammatory diseases (Documents 18 to 21). On the other hand, it is reported that CCR5, which is known as a chemokine CCL2 (MIP-1α), CCL4 (MIP-1β) or CCL5 (RANTES) receptor, is expressed in monocytes and macrophages and participates in various inflammatory diseases similar to CCR2. Furthermore, it is known that CCR2 and CCR5 serve as coreceptors for cell entry of human immunodeficiency virus (HIV) (Deng et al., Nature, 381, 661-666 (1996)). However, a large number of points still remain unknown in intracellular signal transduction cascades relating to leukocyte chemotaxis. Accordingly, there is a great worth in studying the mechanism controlling chemokine receptor-mediated leukocyte chemotaxis.
It is considered that a receptor having an attractant bonded thereto activates a G protein and thus causes the dissociation of the G protein into α- and β-subunits and the formation of a second messenger, thereby initiating actin polymerization and leukocyte chemotaxis (Documents 22 and 23). The dissociation of the G protein is followed by the phosphorylation of the receptor by protein kinases such as PKs, Jaks and GRKs (Documents 24 and 25). Subsequently, the phosphorylation in the intracellular domain in the carboxyl terminal region (C-terminal region) of the receptor promotes receptor internalization with adaptors Aps and arrestin and inhibits excessive responses (Document 26). As the results of conventional studies, there have been known GPCR-binding molecules regulating receptors' functions such as cell-surface transportation and intracellular uptake (Documents 33 and 34). However, there have been known so far few GPCR-binding molecules controlling intracellular chemotactic signal cascades specific to individual receptors.
Studies on mutations have clarified that receptors binding to attaractants such as cAMP, fMLP and chemokines activate chemotactic signal cascades even in the case where phosphorylation does not occur in the intracellular C-terminal domain (Documents 27 to 32). In the previous studies, GPCR-binding molecules, which regulate receptors' functions such as cell-surface transportation and intracellular uptake, were identified (Documents 33 and 34). However, chemotactic signal cascades of individual receptors are scarcely known.
It is reported that the second cytoplasmic loop and the C-terminal domain of a GPCR are important sites in the activation of the chemotactic signal cascade by the binding to a G protein and activation thereof (Documents 27, 28, 35 and 36). When 12 residues in the intracellular C-terminal domain of CCR2 (a sequence in the Pro-12-C terminal domain; SVFFRKHITKRF (SEQ ID NO:41)) is removed, for example, its chemotactic response disappears though the G protein-binding ability to CCL2 remains unchanged (Documents 28 and 37). When a shorter sequence from the terminus is removed, however, no effect is observed. It is interesting that the DRY motif at the second cytoplasmic loop of a chemokine receptor is completely conserved while the sequence in the neighborhood of the terminus is scarcely conserved. Although chemokine receptors CCR2 and CXCR4 both activate monocyte chemotaxis cascades, the Pro-12-C terminal domain of CCR2 never relates to the conservation of a similar domain of CXCR4.